Several scientific or patent publications are referenced in this patent application to describe the state of the art to which the invention pertains. Each of these publications is incorporated by reference herein, in its entirety.
Mammals respond to tissue injury, trauma or infection by executing a complex series of biological reactions in an effort to prevent further tissue damage, to initiate repair of damaged tissue, and to isolate and destroy infective organisms. This process is referred to as the inflammatory response, the early and intermediate stages of which are referred to as the acute phase response.
The acute phase response involves a wide variety of mediators, including cytokines, interleukins and tumor necrosis factor. It also involves a radical alteration in the biosynthetic profile of the liver. Under normal circumstances, the liver synthesizes a range of plasma proteins at steady state concentrations. Some of these proteins, the “acute phase” proteins are induced in the inflammatory response to a level many times greater than levels found under normal conditions. Acute phase proteins are reviewed by Steel & Whitehead (Immunology Today 15: 81-87, 1994).
One of the massively induced acute phase proteins is Serum Amyloid A (SAA). SAA actually comprises a family of polymorphic proteins encoded by many genes in a number of mammalian species. SAAs are small apolipoproteins that accumulate and associate rapidly with high-density lipoprotein 3 (HDL3) during the acute phase of the inflammatory response. Most SAAs are induced in response to inflammation; however, certain SAAs (e.g., human SAA4) appear to be constitutively expressed or minimally induced in the inflammatory response.
SAAs are regulated transcriptionally and post transcriptionally, though transcriptional regulation appears to predominate. SAA mRNA levels have been observed to increase up to 1,000 fold in the hours following an inflammatory stimulus. Likewise, plasma concentrations of SAA protein have been shown to increase as much as 1,000 fold, to levels approaching 1 mg/ml, for short periods following an inflammatory stimulus.
The massive increase in SAA plasma levels in response to both infective and non-infective inflammatory stimuli has led to its use as a diagnostic marker of inflammation. Among the most effective assays are immunoassays utilizing antibodies raised in a species that does not produce detectable amounts of SAA. For instance, McDonald et al. (J. Immunol. Meth. 144: 149-155, 1991) describe an antibody sandwich assay using two purified rat monoclonal antibodies raised against human SAA. Immunoassays utilizing these antibodies were demonstrated to be reliable and sensitive, and do not require denaturation of the specimen prior to assay (McDonald et al., 1991, supra). Similarly, Satoh et al. (Am J. Vet. Res. 56: 1286-1291, 1995) describe an ELISA assay for measuring SAA levels in horse serum using rabbit anti-horse SAA antibodies. Though effective, these and similar immunoassays are invasive in that they require a blood sample. Moreover, they may not be appropriate or effective for early detection of localized inflammation, which is common in connection with a variety of infectious and non-infectious tissue trauma.
One excellent example of a non-systemic inflammatory-related disease of great economic importance to the dairy industry is mastitis. Mastitis is generally regarded as an inflammation of the mammary gland. The disease can affect any mammal, but is most economically significant in dairy heifers and cows. Mastitis usually results from colonization of the mammary gland by pathogenic bacteria. However, physical injuries or local mechanical or chemical stresses in the udder can also trigger a local inflammation cascade without the involvement of any primary bacterial infection (sometimes referred to as sterile mastitis).
Mastitis can be expressed at clinical or subclinical levels, and may be localized to only a portion of the udder. Subclinical or localized mastitis is economically damaging because it often remains undetected and untreated, yet results in decreased milk production. Accordingly, it is important in the early diagnosis of mastitis to be able to detect infection before clinical symptoms arise, and to be able to localize the infection to specific regions of the udder.
Current clinical laboratory methods used for the diagnosis of mastitis include estimation of somatic cell counts (SCC), various electrolyte levels, and soluble proteins, such as lactate dehydrogenase (LDH) and N-acetyl-β-D glucosamidase (NAG), in milk samples, all of which reflect a breakdown in the blood-milk barrier due to the inflammatory response caused by the infection. Certain of these parameters, e.g., SCC and electrolyte estimates, are unable to differentiate infected from uninfected regions of the udder (Zank & Slatterer, J. Vet. Med. 45: 41-51, 1998), while others, e.g., LDH and NAG detection, may not be sufficiently sensitive for very early diagnosis. Accordingly, new indicators that are highly predictive of the onset of mastitis infection at an early stage are needed.